Generally, a method using magnetic particles is widely used in order to isolate a target substance, such as nucleic acid and protein, from a biological sample. In this method, biochemical substances in the suspension state are rapidly attached to the fine magnetic particles having large surface areas, and the magnetic field is applied so as to collect the magnetic particles on which the target substance are attached, and then a solution thereof is removed. And various automatic equipments associated with this method have been developed.
Recently, an automated method using pipettes has been used widely.
U.S. Pat. No. 5,647,994 (claiming a priority date of Jun. 21, 1993) filed by Labsystems Oy describes various methods of separating the magnetic particles using a disposable pipette. These methods are prior arts for collecting the magnetic particles in the pipette like in U.S. Pat. Nos. 5,702,950 and 6,187,270. And the construction elements thereof includes a tubular member serially connected to a jet channel, the jet channel defining a flow port at a free end of the tubular member and also having a diameter that is less than a diameter of a separation chamber; and a magnetic element disposed at one of a first location adjacent to the outer side of the separation wall and a second location within the separation chamber, wherein the magnetic element is adapted to be brought into such a state that magnetic particles will gather under the influence of magnetic field when disposed in a first location, or into such a state that the magnetic field no longer keeps the magnetic particles when disposed in a second location, and wherein the tubular member includes a second portion defining a cylindrical channel serially connected to the separation chamber on a side remote from the jet channel, the cylindrical channel receiving a movable piston thereby defining a suction cylinder for drawing liquid into the separation chamber and for removing the liquid from the separation chamber through the jet channel via the flow port.
A magnetic substance attracting/releasing control method proposed by Precision System Science Co. Ltd (U.S. Pat. Nos. 5,702,950 and 6,231,814) has the same fundamental principle as in U.S. Pat. No. 5,647,994, except that a magnet is attached and detached in one direction of the pipette and thus magnetic field is controlled in one direction of a pipette tip. This magnetic substance attracting/releasing control method comprises the steps of: providing a pipette device having a liquid suction line including a liquid inlet end for sucking liquid containing the magnetic substance from a container and discharging the liquid through the liquid inlet end, and a magnet body or magnet bodies being detachably fitted to an external peripheral surface of the liquid suction line of the pipette device; the pipette device providing attracting/releasing control by absorbing and maintaining the magnetic substance contained in the liquid and attracted to the liquid suction line due to magnetism in the magnet body or bodies on an internal surface of the liquid suction line, the magnetic substance being maintained on the internal surface of the pipette device and also by releasing the magnetic substance from the liquid suction line by means of interrupting effect by magnetism in the magnet body or bodies so that the substance is discharged together with the liquid to outside of the liquid suction line through the liquid inlet end.
In U.S. Pat. No. 6,187,270 (Roche Diagnostics GmbH), there is disclosed a method of separation of magnetic particles, in which a permanent magnet is approached to a disposable tip so as to attach the magnetic particles, thereby separating the magnetic particles from a solution. To this end, an apparatus for separation of the magnetic particles includes a pipette connected to a pump, a magnet, and a means for moving the magnet to the pipette side or the opposite side thereof. According to claim 1 of this patent, it is characterized in that a device for separating magnetic microparticles from a suspension in a liquid comprises a pipette having an inner wall, the pipette containing a suspension of magnetic microparticles in a liquid therein, as pipette contents, wherein the pipette is configured to be rotatable about a longitudinal axis thereof; a pump connected to the pipette; a magnet exterior of the pipette and locatable to apply a magnetic field to at least part of the pipette contents to deposit the microparticles on the inner wall of the pipette; and moving means for causing relative movement of the pipette and the magnet to move at least one of them toward the other. According to claim 2 thereof, it is characterized in that the device comprises a pipette having an inner wall, the pipette containing a suspension of magnetic microparticles in a liquid therein as pipette contents; a pump connected to the pipette; a magnet exterior of the pipette and locatable to apply a magnetic field to at least part of the pipette contents to deposit the microparticles on the inner wall of the pipette; and moving means for causing relative movement of the pipette and the magnet to move at least one of them toward the other, wherein the magnet is configured to be movable around a longitudinal axis of the pipette. In the device of claim 2, the magnet is formed to be movable around the longitudinal axis of the pipette instead of the rotation of the pipette. And according to a third independent claim of this patent, it is characterized in that the pipette and magnet are movable relative to one another so that the magnetic field deposits microparticles on the inner wall in order to collect and eject magnetic microparticles deposited on the inner wall of the pipette in the pipette tip.
In these structures, there have been proposed methods of separating the magnetic particles from a solution using a disposable pipette and then suspending them in other solution. Herein, however, there is a serious problem that a lower end of the pipette may be clogged with the magnetic particles and thus an inaccurate result may be occurred frequently. Further, since a series of processes for separating a target substance from a biochemical mixed liquid is carried out in a pipette, it is difficult to uniformly maintain a suspension of a liquid, and also since alcohol is not completely removed even after a final step of cleaning alcohol, there is another problem that the alcohol is remained upon elution and thus may exert an influence on other post-process such as PCR.
In order to solve these problems, there has been developed again a special method in which a process of catching and collecting magnetic substances from a liquid is performed in a sample vessel instead of a pipette. The method in which a reaction is carried out in the sample vessel had been proposed already before.
Gen-Probe, Inc. also discloses a method of separating magnetically attractable particles from a fluid using a magnetic separation rack. The magnetic separation rack includes a plurality of test tube retaining portions, and a plurality of magnets arranged such that each magnet is alternately located at one side of the test tube. Herein, the magnet is arranged to one side of the test tube and at an upper portion thereof but not contacted with a bottom surface thereof. The magnetically labeled particles are attracted to the side of the test tube and then rinsed or removed with a liquid, thereby performing quantitative analysis thereof. However, since the magnetic particles in the test tube cannot sensitively response to the magnetic separation rack, there is a disadvantage in that the purification efficiency is deteriorated.
Amersham International plc had developed a method which can switch a magnet filed by moving a doughnut-shaped magnet vertically to a vessel (U.S. Pat. No. 5,897,783). And Beckman instruments, Inc. had developed an automatic purification apparatus for separating a magnetic substance from a sample using a magnetic plate (EP0479448). The magnetic plate is formed into a plate having a plurality holes through which a vessel can be inserted, and the automatic purification apparatus includes meaning for moving the magnetic plate up and down.
In case that the magnet has to be manufactured according to a certain shape and size, such as a doughnut and a plate, it is difficult to manufacture it and the manufacturing cost is increased. Thus, it is economically infeasible.
In case of using a commercially available rod magnet, it is advantageous to fabricate the apparatus. Gene-Trak systems had proposed a magnetic separation device using such the rod magnet (EP0317286). The magnetic separation apparatus includes base means having a plurality of orifices for receiving nonferrous containers adapted to contain magnetic particles; and a plurality of magnet means mounted on the said base spaced about the periphery of each receiving orifice wherein each of the said magnet means possess a north-south magnetic field orientation in a direction which is coplanar with a cross-sectional plane through the receiving orifices and wherein each of the north-south magnet fields of the magnet means are oriented in a common direction. However, in this system, there is not disclosed a method of minutely controlling up and down movements of the magnet means.
Meanwhile, temperature controlling in purification of a biological sample is one of the very important parameters. The optimal active temperature of protease K which is generally applied upon lysis is 55˜65, and the temperature controlling is essential in the purification. Further, after the final step of cleaning alcohol, it is preferable to completely dry and remove the alcohol. If the alcohol is not completely removed, other post-process such as PCR is influenced by the alcohol remained even after elution. Therefore, it is necessary to perform a high temperature treatment, thereby completely removing the alcohol.
Nevertheless, in the existing automatic purification apparatuses, temperature controlling means for improving the purification efficiency is not considered yet. Generally, the existing automatic systems which have been developed until now do not treat the lysis step at all which needs the temperature controlling. Even though the temperature controlling is carried out, the sample has to be moved to a new block in which the temperature controlling can be performed and then reacted therein. However, in this case, loss of the sample is inevitable due to the frequent movement of the sample.
Furthermore, in protein expression outside of cells, it is essential to control the temperature into 30˜40° C. and properly maintain the temperature for 3 hours or more. In a test tube, enzyme activity has to be occurred in all processes that RNA is synthesized from DNA and a protein is synthesized from the RNA. Herein, the above-mentioned reactions are carried out only when the temperature is maintained into 30˜40° C.
Also in case of using the above-mentioned pipettes, it is actually impossible to control the temperature due to its structural problem.
Therefore, in order to purify the biological sample using the magnetic particles, there is an increasing necessity for a new automatic purification apparatus in which the magnetic field is applied to the outside of a test tube, not a pipette and its up and down movement and temperature can be controlled and also which can be facilely fabricated.